The current invention relates to new HIV-1 group O antigens, nucleic acids encoding them, and the use of said antigens and/or nucleic acids as reagents in the diagnosis and prophylaxes of AIDS. It also relates to new HIV-1 group O strains comprising these antigens.
The human immunodeficiency virus (HIV) is the responsible agent for the acquired immunodeficiency syndrome (AIDS) in humans. AIDS is usually associated with two distinct types of HIV: HIV-1 and HIV-2, initially described by Gallo et al. (1984) and Barrxc3xa9-Sinoussi et al. (1983) on the one hand, and Clavel et al. (1986) on the other hand. Although both types, HIV-1 and HIV-2, cause a dysfunction of the immune system and induce similar clinical symptoms in infected persons, they are genetically distinct (Clavel et al. 1986) Epidemiological studies have shown that the prevalence of HIV-2 infection is confined mainly to West Africa, whereas HIV-1 infection is a world wide problem. Numerous HIV-1 isolates have been obtained and sequenced from diverse geographical locations. At present, at least ten distinct subgroups or clades (A to J) of HIV-1 have been described, equidistantly related in phylogenetic analysis of the env-and/or gag-gene (Kostrikis et al. 1995; Louwagie et al. 1993; Myers et al 1995).
More recently, HIV-1 group O (for xe2x80x9cOutlierxe2x80x9d) strains have been described as divergent viruses, belonging to an independent cluster (Charneau et al. 1994; Gxc3xcrtler et al. 1994; Myers et al 1995; Sharp et al. 1994; Vanden Haesevelde et al. 1996), when compared to the vast majority of worldwide HIV-1 strains classified as group M (for xe2x80x9cMajorxe2x80x9d). Although these two groups of viruses share the same genomic structure, the elevated level of divergence between them supports the hypothesis of independent origins.
Most of the currently described group O strains have been characterized from Cameroonian patients or from patients who have travelled in Cameroon (De Leys et al. 1990; Gxc3xcrtler et al. 1994; Loussert-Ajaka et al. 1995; Vanden Haesevelde et al. 1996). Group O infection is not restricted to Cameroon and its neighbouring countries, but it has also been documented in West, East, and Southern Africa (Peeters et al. 1996; Peeters et al. submitted). In addition, cases of group O infection have been described in several European countries (France, Spain, Germany, Norway) and in the USA (Centres for Disease control and Prevention 1996; Charneau et al. 1994; Hampl et al 1995; Soriano et al. 1996).
Several hypotheses have been developed to explain the paradoxical observation that HIV-1 has been present in African countries for many decades (probably about a century) and that it has only become apparent over the past 15 years. The answer should probably take in account numerous parameters such as demographic, sociologic, ethologic, ethnologic, and virologic parameters. In a mathematical model, May and Anderson (1990) suggest that initial chains of infection were found in isolated populations at low rates with some xe2x80x98sparksxe2x80x99 thrown in the neighbouring villages, and the exponential epidemic has started when there was a sufficient number of fire-boxes. To date, no differences were observed between HIV-1 group M and O pathogenic potential even though a limited number of patients infected by these latter strains have been reported. However some of them have already died or reached stage IV in the CDC classification (Charneau et al. 1994; Gxc3xcrtler et al. 1994; Loussert-Ajaka et al. 1995). It is possible that group O epidemics, compared to group M, could be rampant at this time. In the next years, it will therefore be extremely important to monitor the prevalence of these viruses, in Africa but also in the developed countries, to detect them as early as possible and to prevent a new HIV epidemic.
HIV-1 group O strains present a public health challenge since they are documented to give incomplete and atypical HIV-1 Western blot profiles (Charneau et al. 1994; Gxc3xcrtler et al. 1994). Some commercially available ELISA or rapid tests were unable to detect HIV-antibodies in HIV-1 group O infected patients (Loussert-Ajaka et al 1994; Simon et al. 1994). The distribution of group O infections may be much more wide spread than currently thought, because of a lack of adequate detection techniques. Moreover, whereas HIV-1 group M strains have been extensively studied and characterized as to their genetic variability, there is at present no clear view on the genetic diversity of strains belonging to HIV-1 group O.
At present, sequence information on the complete genome is only available for the prototype isolates of HIV-1 group O, namely ANT70 (Vanden Haesevelde et al. 1994), MVP-5180 (Gxc3xcrtler et al. 1994), and VAU (Charneau et al. 1994). Some additional HIV-1 group O strains have been sequenced in the gag and env regions (for example WO 96/27013, WO 96/12809, EP 0727483).
HIV-viruses show a high degree of genetic variability. In the case of HIV-1 viruses it is more or less accepted that at least one nucleotide change occurs during one replication cycle. Certain regions of the genome, for example those encoding structurally or enzymatically important proteins, may be rather conserved, but other regions, especially the env-region, may be subject of very high genetic variability.
The envelope proteins of HIV are the viral proteins most accessible to immune attack, and much attention has been directed towards elucidating their structure and function. The env gene encoding the envelope proteins consists of hypervariable sequences (V-regions) alternated by more constant regions (C-regions) (Starcich et al, 1986; Willey et al, 1986). The envelope protein is first synthesized as a heavily glycosylated precursor protein (gp160), which is later cleaved by a non-viral protease to generate a transmembrane protein, also referred to as gp41, and an outer surface protein often referred to as gp120. One particular region of the gp120 glycoprotein derived from the HIV-1 virus type has been studied extensively, namely the third hypervariable domain (V3) also known as the principal neutralizing determinant (PND) (Javaherian et al., 1989). The V3 domain of HIV-1 contains a loop structure of 35 amino acids (V3-loop) which is formed by a cysteine-cysteine disulfide bridge (Leonard et al. 1990). The gp41 protein contains an immunodominant domain (ID) as found in all retroviruses. For HIV-viruses, this domain has been divided in two distinct regions, corresponding to an immunosuppressive peptide (ISU) of about 17 aa, and a cysteine loop being the principal immunodominant domain (PU)). The delineation of these respective regions in the gp41 protein is demonstrated in FIG. 1.
The genetic variability of HIV-viruses considerably complicates both diagnosis and prevention of HIV-infection. Sera from patients infected with unknown types of HIV-virus, may contain antibodies which are not detected by the current assay methods, which are based on (poly)peptide sequences of known viral strains. The detection of virus or viral antigen in certain samples, like organs for transplantation, or blood transfusion samples, may be missed due to the presence of hitherto unknown variant types. Variation may occur in those genomic regions which are considered to be important in future vaccines. Finally, it is not known at present if different genoric types may influence the course of the AIDS disease, i.e. its virulence and/or susceptibility for therapeutics.
Therefore, there is a constant need for characterization and sequencing of new HIV-strains, and especially of new HIV-1 group O strains, which until now have only scarcely been characterized. Information on the genetic variability of this xe2x80x9cOutlierxe2x80x9d group may enable a more rational approach for optimization of diagnostic tests and for development of vaccines. Especially the variability of certain regions in the genome, known to be important target regions for the immune response, or for certain therapeutic drugs, is of utmost importance. New sequencing data may require the revision of existing diagnostic assays, and/or the development of new assays. Depending on the situation, it may be important to obtain a general detection of all HIV-infected samples, with a low number of false positives and false negatives, or to be able to differentiate different types of HIV-infection (such as HIV-1 group M, HIV-1 group O, HIV-2).
It is the aim of the current invention to provide new nucleic acid and peptide sequences originating from HIV-1 group O strains.
It is more specifically the aim of the current invention to provide nucleic acid and peptide sequences corresponding to the env-region of new HIV-1 group O strains, more particularly corresponding to the gp160 env-precursor protein region, and most particularly to the C2V3 region and the gp41 region.
It is also an aim of the present invention to provide for new viral strains belonging to HIV-1 group O.
It is moreover an aim of the present invention to provide for antigens derived from said new HIV-1 group O strains.
It is also an aim of the current invention to provide for nucleic acids derived from said new HIV-1 group O strains.
It is also an aim of the present invention to provide antibodies reacting specifically with the antigens from the new HIV-1 group O strains.
It is moreover an aim of the present invention to provide for probes hybridizing specifically with the nucleic acids of the new HIV-1 group O strains.
It is moreover an aim of the present invention to use said antigens and/or antibodies and/or probes in a test for detecting the presence of HIV-infection and/or to differentiate different types of HIV-infection.
It is thus also an aim of the present invention to provide for assays enabling the detection and/or differentiation of HIV-infections.
It is finally also an aim of the present invention to provide for vaccine compositions providing protection against AIDS.
The following definitions serve to illustrate the terms and expressions used in the different embodiments of the present invention as set out below:
The term xe2x80x9cpolynucleic acidxe2x80x9d corresponds to either double-stranded or single-stranded cDNA or genomic DNA or RNA, containing at least 10, 20, 30, 40 or 50 contiguous nucleotides. Single stranded polynucleic acid sequences are always represented in the current invention from the 5xe2x80x2 end to the 3xe2x80x2 end.
Polynucleic acids according to the invention may be prepared by any method known in the art for preparing polynucleic acids (e.g. the phosphodiester method for synthesizing oligonucleotides as described by Agarwal et al. (1972), the phosphotriester method of Hsiung et al. (1979), or the automated diethylphosphoroamidite method of Baeucage et al. (1981)). Alternatively, the polynucleic acids of the invention may be isolated fragments of naturally occurring or cloned DNA, cDNA or RNA.
The term xe2x80x9coligonucleotidexe2x80x9d refers to a single stranded nucleic acid comprising two or more nucleotides, and less than 100 nucleotides. The exact size of an oligonucleotide depends on the ultimate function or use of said oligonucleotide. For use as a probe or primer the oligonucleotides are preferably about 5-50 nucleotides long, more preferably 10-30 nucleotides long.
The oligonucleotides according to the present invention can be formed by cloning of recombinant plasmids containing inserts including the corresponding nucleotide sequences, if need be by cleaving the latter out from the cloned plasmids upon using the adequate nucleases and recovering them, e.g. by fractionation according to molecular weight. The probes according to the present invention can also be synthesized chemically, e.g. by automatic synthesis on commercial instruments sold by a variety of manufacturers.
The nucleotides as used in the present invention may be ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine or nucleotides containing modified groups which do not essentially alter their hybridisation characteristics. Moreover, it is obvious to the man skilled in the art that any of the below-specified probes can be used as such, or in their complementary form, or in their RNA form (wherein T is replaced by U).
The oligonucleotides used as primers or probes may also comprise or consist of nucleotide analogues such as phosphorothioates (Matsukcura et al., 1987), alkylphosphorothioiates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or may contain intercalating agents (Asseline et al., 1984).
As most other variations or modifications introduced into the original DNA sequences of the invention, these variations will necessitate adaptions with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity. However the eventual results of the hybridisation or amplification will be essentially the same as those obtained with the unmodified oligonucleotides.
The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridization kinetics, reversibility of the hybrid-formation, biological stability of the oligonucleotide molecules, immobilization to solid phase etc.
The term xe2x80x9cprobexe2x80x9d refers to single stranded sequence-specific oligonucleotides which have a sequence which is sufficiently complementary to hybridize to the target sequence to be detected.
Preferably said probes are 90%, 95% or more homologous to the exact complement of the target sequence to be detected. These target sequences may be genornic DNA, genomic RNA or messenger RNA, or amplified versions thereof.
The term xe2x80x9chybridizes toxe2x80x9d refers to preferably stringent hybridization conditions, allowing hybridisation between sequences showing at least 90%, 95% or more homology with each other.
The term xe2x80x9cprimerxe2x80x9d refers to a single stranded DNA oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products. Preferably the primer is about 5-50 nucleotides long. Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength. The fact that amplification primers do not have to match exactly with the corresponding template sequence to warrant proper amplification is amply documented in the literature (Kwok et al., 1990).
The amplification method used can be either polymerase chain reaction (PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu and Wallace, 1989, Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990, Compton, 1991), transcription-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992) or amplification by means of Qxcex2 replicase (Lizardi et al., 1988; Lomeli et al., 1989) or any other suitable method to amplify nucleic acid molecules.
The term xe2x80x9ccomplementaryxe2x80x9d nucleic acids as used in the current invention means that the nucleic acid sequences can form a perfect base paired double helix with each other.
The terms xe2x80x9cpolypeptidexe2x80x9d and xe2x80x9cpeptidexe2x80x9d are used interchangeably throughout the specification and designate a linear series of amino acids connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acids. Polypeptides can be of a variety of lengths, either in their natural (uncharged) forms or in a charged form (=salt form), and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications. Preferably the peptides of the invention are less than 100 amino acids in length, more preferably less than 50, and even less than 30 amino acids long. It is well understood in the art that amino acid sequences contain acidic and basic groups, and that the particular ionization state exhibited by the peptide is dependent on the pH of the surrounding medium when the protein is in solution, or that of the medium from which it was obtained if the protein is in solid form. Also included in the definition are proteins modified by additional substituents attached to the amino acids side chains, such as glycosyl units, lipids, or inorganic ions such as phosphates, as well as modifications relating to chemical conversions of the chains, such as oxidation of sulfhydryl groups. Thus, xe2x80x9cpolypeptidexe2x80x9d or its equivalent terms is intended to include the appropriate amino acid sequence referenced, subject to those of the foregoing modifications which do not destroy its functionality.
The polypeptides of the invention, and particularly the fragments, can be prepared by classical chemical synthesis.
The synthesis can be carried out in homogeneous solution or in solid phase.
For instance, the synthesis technique in homogeneous solution which can be used is the one described by Houbenweyl in the book entitled xe2x80x9cMethode der organischen chemiexe2x80x9d (Method of organic chemistry) edited by E. Wunsh, vol. 15-I et II. THIEME, Stuttgart 1974.
The polypeptides of the invention can also be prepared in solid phase according to the methods described by Atherton and Shepard in their book entitled xe2x80x9cSolid phase peptide synthesisxe2x80x9d (IRL Press, Oxford, 1989).
The polypeptides according to this invention can also be prepared by means of recombinant DNA techniques as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory, 1982). In that case the polypeptides are obtained as expression products of the nucleic acids encoding said polypeptides. The expression occurs in a suitable host cell (eukaryotic or prokaryotic) which has been transformed with a vector in which the nucleic acid encoding the polypeptide has been inserted (called xe2x80x9cinsertxe2x80x9d). The nucleic acid insert may have been obtained through classical genomic cloning techniques (screening of genomic libraries, shotgun cloning etc . . . ), or by amplification of the relevant part in the viral genome, using suitable primer pairs and, for example, the polymerase chain reaction, or by DNA synthesis.
The word xe2x80x9cantigenxe2x80x9d refers to a molecule which provokes an immune response (also called xe2x80x9cimmunogenxe2x80x9d), or which can be recognized by the immune system (also called xe2x80x9cantigen sensu strictuxe2x80x9d). The immune response or the immune recognition reaction can be of the cellular or humoral type. The antigens of the current invention are all polypeptides or peptides, and therefore, the words xe2x80x9cantigenxe2x80x9d and xe2x80x9c(poly)peptidexe2x80x9d may be used interchangeably throughout the current invention.
The term xe2x80x9cantigenic determinantxe2x80x9d or xe2x80x9cepitopexe2x80x9d refers to that portion of an antigenic molecule that is specifically bound by an antibody combining site. Epitopes may be determined by any of the techniques known in the art or may be predicted by a variety of computer prediction models known from the art.
The terms xe2x80x9chomologousxe2x80x9d and xe2x80x9chomologyxe2x80x9d are used in the current invention as synonyms for xe2x80x9cidenticalxe2x80x9d and xe2x80x9cidentityxe2x80x9d; this means that amino acid sequences which are e.g. said to be 55% homologous, show 55% identical amino acids in the same position upon alignment of the sequences. The same definition holds for homologous nucleic acid sequences, i.e. nucleic acid sequences which are e.g. said to be 55% homologous, show 55% identical base pairs in the same position upon alignment of the sequences.
The aims of the present invention have been met by the following embodiments.
The present invention provides for an antigen, derived from the gp160-env precursor protein of a new HIV-1 group O strain, and characterized by an amino acid sequence comprising at least one of the following sequences:
VQQMKI (SEQ ID NO 53),
KIGPMSWYSMG (SEQ ID NO 54),
GLEKN (SEQ ID NO 55),
IQQMKI (SEQ ID NO 56),
KIGPLAWYSMG (SEQ ID NO 57),
MGLERN (SEQ ID NO 58),
QSVQEIKI (SEQ ID NO 59),
KIGPMAWYSIG (SEQ ID NO 60),
IGIGTT (SEQ ID NO 61),
VQEIQT (SEQ ID NO 62),
QTGPMAWYSIH (SEQ ID NO 63),
IHLRTP (SEQ ID NO 64),
IQEIKI (SEQ ID NO 65),
KIGPMSWYSMG (SEQ ID NO 66),
MGIGQE (SEQ ID NO 67),
SVQELRI (SEQ ID NO 68),
RIGPMAWYSMT (SEQ ID NO 69),
MTLERD (SEQ ID NO 70),
SVQEIPI (SEQ ID NO 136),
and/or at least one amino acid sequence chosen from the following group of sequences
RNQQLLNLWGCKGRLIC (SEQ ID NO 71),
CKGRLICYTSVQWNM (SEQ ID NO 72),
LWGCKGRIVC (SEQ ID NO 73),
SLWGCKGKLIC (SEQ ID NO 74),
CKGKSIC (SEQ ID NO 75),
CKGKIVC (SEQ ID NO 76),
CRGRQVC (SEQ ID NO 77),
CKGRLICYTSVH (SEQ ID NO 79),
CKGNLIC (SEQ ID NO 80),
CKGKMIC (SEQ ID NO 81),
CKGRVVC (SEQ ID NO 82),
or a fragment of said antigen, said fragment consisting of at least 8, preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous amino acids of the amino acid sequence of said antigen, and being characterized by the fact that it specifically reacts with antibodies raised against said antigen.
The term xe2x80x9cderived fromxe2x80x9d signifies that the antigen contains a fragment of the gp160 env precursor protein.
The expression xe2x80x9cspecifically reacts withxe2x80x9d means that the antigen fragment is specifically recognized by antibodies raised against the antigen from which it is derived. Specificity of reaction may be preferably demonstrated using monoclonal antibodies raised against the antigen of the invention. Specificity of polyclonal antibodies may be obtained after absorption of said antibodies with the corresponding antigens of other HIV-1 group O strains, in order to eliminate non-specific antibodies (=cross reactive antibodies) present in the polyclonal mixture. The expression xe2x80x9cspecifically react withxe2x80x9d also means that sera taken from patients infected with the HIV-1 group O strain from which the antigen of the invention originates, show a preferential reaction with the antigen or antigen fragment of the invention, as compared to the reactivity with a corresponding antigen or antigen fragment of other HIV-1 group O strains (=control), under comparable reaction conditions. This preferential reaction may be measured quantitatively (e.g. ELISA absorption values) and should result in reactivity values which are at least 20%, 30%, 40% and preferably 50% higher than the reactivity with the control antigen. In practice, this means that the selected fragments of the above-mentioned antigens will always show at least one amino acid difference when compared in an alignment with the sequence of corresponding antigens of other HIV-1 group O isolates, such as ANT70, MVP5180, VAU or others.
The above-mentioned amino acid sequences SEQ ID NO 53 to 70 and 136 originate from the central region in the V3 loop of the gp160-env precursor protein of new HIV-1 group O strains, while the amino acid sequences represented by SEQ ID NO 71-77 and 79-82 originate from the gp41-principal immunodominant domain (PID) of the gp160-env precursor protein of the same HIV-1 group O strains.
The current invention also provides for antigens consisting of any of the amino acid sequences represented by SEQ ID NO 53-70, 136, 71-77, 79-82, or consisting of an amino acid sequence according to any of SEQ ID NO 53-70, 136, 71-77, 79-82, whereby said sequence is extended at its N-terminal and/or C-terminal end with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids.
The invention further provides for an antigen as described above, characterized by an amino acid sequence comprising at least one of the following amino acid sequences:
CERPGNNSIQQMKIGPLAWYSMGLERNKSSISRLAYC (SEQ ID NO 83),
CERPGNNSIQQMKIGPMAWYSMGLERNKSSISRLAYC (SEQ ID NO 84),
CERPGNQSVQEIKIGPMAWYSIGIGTTPANWSRIAYC (SEQ ID NO 85),
CERPGNQSVQEIKIGPMAWYSIGIGTTPTYNWSRIAYC (SEQ ID NO 86),
CVRPWNQTVQEIQTGPMAWYSIHLRTPLANLSRIAYC (SEQ ID NO 87),
CQRPGNLTIQEIKIGPMSWYSMGIGQEDHSKSRNAYC (SEQ ID NO 88),
CERPYYQSVQELRIGPMAWYSMTLERDRAGSDIRAAYC (SEQ ID NO 89),
CERPGNHTVQQMKIGPMSWYSMGLEKNNTSSRRAFC (SEQ ID NO 90),
CERTWNQSVQEIPIGPMAWYSMSVELDLNTTGSRSADC (SEQ ID NO 135),
and/or at least one amino acid sequence chosen from the following group of sequences:
DQQLLNLWGCKGRIVCYTSVKWN (SEQ ID NO 91),
NQQLLNLWGCKGRLVCYTSVKWNK (SEQ ID NO 92),
NQQLLNLWGCKGRLVCYTSVKWNN (SEQ ID NO 138),
NQQRLNLWGCKGKMICYTSVPWN (SEQ ID NO 93),
NQQLLNLWGCKGKSICYTSVKWN (SEQ ID NO 94),
NQQLLNLWGCKGRLICYTSVQWN (SEQ ID NO 95),
NQQRLNLWGCKGKMICYTSVKWN (SEQ ID NO 96),
NQQLLNLWGCKGNLICYTSVKWN (SEQ ID NO 97),
NQQLLNLWGCRGRQVCYTSVIWN (SEQ ID NO 98),
SQQLLNLWGCKGRLICYTSVHWN (SEQ ID NO 99),
NQQLLNLWGCKGRIVCYTSVKWN (SEQ ID NO 100),
NQQLLNSWGCKGKIVCYTAVKWN (SEQ ID NO 101),
NQQLLSLWGCKGKLICYTSVKWN (SEQ ID NO 102),
NQQLLNLWGCKGRLVCYTSVQWN (SEQ ID NO 137),
or a fragment of said antigen, said fragment consisting of at least 8, preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous amino acids of the amino acid sequence of said antigen, and being characterized by the fact that it specifically reacts with antibodies raised against said antigen.
The above-mentioned amino acid sequences SEQ ID NO 83 to 90 and 135 represent the V3 loop region of the gp160-env precursor protein of new HIV-1 group O strains, while the amino acid sequences SEQ ID NO 91 to 102, 137 and 138 originate from the gp41-immunodominant domain (ID) of the gp160-env precursor protein of the same HIV-1 group O strains.
The current invention also provides for antigens consisting of any of the amino acid sequences represented by SEQ ID NO 83-102, 135, 137 and 138 or consisting of an amino acid sequence according to any of SEQ ID NO 83-102, 135, 137 and 138, whereby said sequence is extended at its N-terminal and/or C-terminal end with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids.
The invention further provides for antigens as above-defined, characterized by an amino acid sequence comprising at least one of the amino acid sequences represented by SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40 as shown in the alignment on FIG. 1, and/or at least one of the amino acid sequences represented by SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, or SEQ ID NO 52 as shown in the alignment on FIG. 2, and/or the amino acid sequence represented by SEQ ID NO 134, or a fragment of said antigen, said fragment consisting of at least 8, preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous amino acids of any of the sequences represented by SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, or SEQ ID NO 134, with said antigen fragment characterized by the fact that it specifically reacts with antibodies raised against the antigen from which it is derived.
Furthermore, the invention provides for an antigen as above-defined, characterized by an amino acid sequence consisting of at least one of the following sequences: SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, or the amino acid sequence represented by SEQ ID NO 134 or a fragment of said antigen, said fragment consisting of at least 8, preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous amino acids of any of the sequences represented by SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, or SEQ ID NO 134, with said antigen fragment characterized by the fact that it specifically reacts with antibodies raised against the antigen from which it is derived.
It is to be noted that all the above-mentioned amino acid sequences originate from HIV-1 group O strains, which have until now never been described. More particularly, as is shown further in the examples section, the new amino acid sequences originate from the following strains:
The amino acid sequences represented by SEQ ID NO 2, 4, 42, 73, 59, 60, 61, 73, 85, 86, 100 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed MP340, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 6, 8, 44, 56, 57, 58, 82, 83, 84, 138 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed FABA, or alternatively termed MP331, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 10, 12, 46, 62, 63, 64, 73, 87, 100 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed MP450, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 14, 16, 48, 65, 66, 67, 76, 88, 101 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed MP448, or a quasi species thereof.
The amino acid sequences represented by SEQ ID NO 18, 50, 53, 54, 55, 73, 90, 91 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed 189, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 40, 52, 68, 69, 70, 71, 89, 95 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed MP539, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 20 and 92 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed 320, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 22, 80 and 97 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed BSD422, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 24, 79 and 99 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed KGT008, or a quasi-species thereof.
The amino acid sequence represented by SEQ ID NO 26 originates from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed MP575, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 28, 72 and 95 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed BSD189, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 30, 77 and 98 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed BSD649, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 32, 81 and 96 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed BSD242, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 34, 81 and 93 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed 533, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 36, 75 and 94 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed 772P94, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 38, 74 and 102 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed MP95B, or a quasi-species thereof.
The amino acid sequences represented by SEQ ID NO 134, 135, 136, and 137 originate from the gp160 env precursor antigen isolated from a HIV-1 group O strain termed MP645, or a quasi-species thereof.
It is noted that the amino acid sequence represented by SEQ ID NO 73 is characteristic for the gp41 immunodominant region of at least the following new HIV-1 group O strains: MP340, MP450, and 189.
The current invention therefore specifically relates to env-derived antigens comprising the characteristic sequence represented by SEQ ID NO 73, as well as virus strains containing these antigens.
It is also noted that the amino acid sequence represented by SEQ ID NO 81 is characteristic for the gp41 immunodominant region of at least the following new HIV-1 group O strains: BSD242 and 533.
The current invention therefore specifically relates to env-derived antigens comprising the characteristic sequence represented by SEQ ID NO 81, as well as virus strains containing these antigens.
It is also noted that the amino acid sequence represented by SEQ ID NO 95 is characteristic for the gp41 immunodominant region of at least the following new HIV-1 group O strains: MP539 and BSD189.
The current invention therefore specifically relates to env-derived antigens comprising the characteristic sequence represented by SEQ ID NO 95, as well as virus strains containing these antigens.
The term xe2x80x9cquasi-speciesxe2x80x9d refers in general to the group of related but genetically and possibly biologically different viruses (also called xe2x80x9cvariantsxe2x80x9d) that an infected individual harbors. The term xe2x80x9crelatedxe2x80x9d means that the xe2x80x9cvariantsxe2x80x9d all arise from a single infectious agent, in this case from a single HIV-1 group O strain. It has been calculated that an HIV-infected patient carries about 106 to 108 genetically distinct HIV-variants, which are generated by the high error rate of reverse transcriptase and the high turnover rate in vivo. In the context of the current description the term xe2x80x9cquasi-speciesxe2x80x9d refers also to a strain isolated from the quasi-species xe2x80x9cgroupxe2x80x9d as above-defined.
The term xe2x80x9cgenetically differentxe2x80x9d means that the nucleic acid sequence of the genome of one strain shows at least one nucleotide difference with the corresponding sequence of another strain belonging to the same quasi-species.
The term xe2x80x9cbiologically differentxe2x80x9d means that some strains of a quasi-species may have different biological characteristics compared to the biological characteristics of other strains from the same quasi-species. These biological characteristics may encompass for example the HIV-1 cell tropism, viral virulence, the capacity to induce syncytia, etc.
Nucleic acid sequences originating from quasi-species differ from each other but always show a high percentage of homology, most often a homology of 90%, 95% or higher. The same holds for the sequence of polypeptides originating from quasi-species. Homology percentages on the protein level usually exceed 95%, 96%, 97%, 98%, or even 99%. These percentages of homology count for the comparison of sequence stretches which are at least 100 nucleotides (about 33 amino acids), and preferably 200, 300 or more nucleotides long (66, 100 or more amino acids). It has to be understood that, when very short sequence stretches are compared (e.g. stretches of about 30 nucleotides, or 10 amino acids) the homology ranges may be much lower, if these short sequence stretches contain the mutual differences.
Examples of sequences originating from xe2x80x9cquasi-speciesxe2x80x9d are provided further in the examples section, where gp41- and C2V3-nucleotide and amino acid sequences of certain strains belonging to the same xe2x80x9cquasi-speciesxe2x80x9d are compared to each other. For example, for strains MP340, FABA, MP450 and MP448 gp41-nucleic acid sequences have been determined on different samples, originating from the same patient, i.e. on serum samples and on peripheral blood monocyte (PBMC) samples. Table 2 shows that, in these specific examples, homology percentages vary from 95% to 100% between gp41-nucleic acid sequences determined on serum samples as compared to PBMC-samples.
It is to be understood that the amino acid and nucleic acid sequences of the current invention also encompass those sequences which are not explicitly recited, but which have been determined on xe2x80x9cquasi-speciesxe2x80x9d of the respective viral strains. As indicated above, these xe2x80x9cvariantxe2x80x9d sequences show a homology range of at least 90%, preferably 95% with the sequences which are specifically recited in the current application.
The above-mentioned antigens are polypeptide or peptide molecules, which are characterized by the above-mentioned amino acid sequences. It has to be understood however, that these (poly)peptides may be modified by for example glycosylation, side chain oxidation or phosphorylation as explained above. A very particular type of side chain oxidation is cyclisation by bridge formation between the xe2x80x94SH groups of two cysteine residues in the same (poly)peptide chain. The cyclic (poly)peptides formed in this way by Sxe2x80x94S bridging may be particularly suitable to expose epitopes located in the loop structure. Epitopes presented in this manner may be in a better shape to be recognized by the immune system, and more particularly by antibodies possibly present in the serum of HIV-infected persons.
A preferential embodiment of the current invention provides for any of the above-mentioned (poly)peptides in a cyclic form.
Cyclisation may occur between two cysteine residues which are present in the above-cited amino acid sequences. For example, cyclic peptides with a loop structure of about 6 amino acids long may be formed with the amino acid sequences represented by e.g. SEQ ID NO 71 to 82, and 91 to 102, and 137. Another example are the V3-loop peptides of about 35 amino acids long, which may be formed by cyclisation of the cysteine residues of the amino acid sequences represented by e.g. SEQ ID NO 83 to 90, and 135.
On the other hand, cyclisation may also be induced in amino acid sequences which do not contain two cysteine residues naturally, but which have been extended with one or two cysteine residues at their extremities, or at in internal position inside the amino acid chain. The current invention therefore also refers to (poly)peptides characterized by any of the above-mentioned amino acid sequences, modified by addition of one or several cysteine residues, at the C-terminal and/or N-terminal extremity and/or inside the (poly)peptide chain.
Another particular type of modification includes the extension of the N-terminal and/or C-terminal end of the (poly)peptide antigen by linker sequences, said linker sequences comprising for example additional amino acids or other molecules (such as for example biotin). The addition of linker sequences to the polypeptide antigen may have several advantages such as:
a more efficient immobilisation on a solid substrate,
a more efficient presentation of the immunoreactive epitope(s) in the (poly)peptide,
linkage to other antigenic determinants . . . .
A preferential embodiment therefore includes antigens or antigen fragments comprising any of the above-mentioned amino acid sequences, extended with linker sequences.
It has to be understood that the above-mentioned (poly)peptide antigens of the invention may be prepared by different methods known in the art. They may be prepared by synthetic means as described above, or they may be produced by recombinant DNA technology. In the latter case, they are the result of the expression of the nucleic acids encoding said antigens or antigen fragments in an appropriate host cell.
The invention also relates to a recombinant vector for the expression of any of the above-mentioned polypeptides, recombinant host cells expresssing these polypeptides, and processes for the recombinant expression of these polypeptides; said tools for recombinant expression are well known by anyone skilled in the art, and have been described in more detail for example in WO96/13590.
The invention further provides for a (poly)nucleic acid encoding any of the above-mentioned (poly)peptide antigens.
More particularly, the current invention provides for a polynucleic acid comprising a nucleotide sequence chosen from the group of
(I) a nucleotide sequence represented by SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, or SEQ ID NO 51, SEQ ID NO 106 or
(ii) a nucleotide sequence complementary to a sequence according to (I), or
(iii) a nucleotide sequence showing at least 95%, preferably 96%, 97%, 98% and most preferably 99% homology to a sequence according to (I), or
(iv) a nucleotide sequence according to (I) whereby T is replaced by U, or
(v) a nucleotide sequence according to (I) whereby at least one nucleotide is substituted by a nucleotide analogue.
It is to be noted that, as will be shown further on in the examples section, the above-mentioned polynucleic acids all originate from the env-gene of new HIV-1 group O strains. The nucleic acid sequences represented by SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39 correspond to the region encoding the gp41-immunodominant domain in the env-gene, while the nucleic acid sequences represented by SEQ ID NO 41, 43, 45, 47, 49, and 51 correspond to the region encoding the C2V3 region in this same env-gene. The nucleotide sequence represented by SEQ ID NO 106 is illustrated in FIG. 8A, and comprises the full env-gene of a new HIV-1 group O strain, termed MP645, together with additional accompanying genes.
The nucleotide sequences mentioned above under item (iii) represent variant nucleic acid sequences which may be isolated e.g. from strains belonging to the same quasi-species.
More particularly, the invention provides for a polynucleic acid consisting of a nucleotide sequence chosen from the group of
(I) a nucleotide sequence represented by SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, or SEQ ID NO 51, SEQ ID NO 106 or
(ii) a nucleotide sequence complementary to a sequence according to (I), or
(iii) a nucleotide sequence showing at least 95%, preferably 96%, 97%, 98% and most preferably 99% homology to a sequence according to (I), or
(iv) a nucleotide sequence according to (I) whereby T is replaced by U, or
(v) a nucleotide sequence according to (I) whereby at least one nucleotide is substituted by a nucleotide analogue.
The invention further provides for a nucleic acid fragment consisting of a sequence of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of the sequence of a polynucleic acid as specified above, and characterized by the fact that it selectively hybridizes to the polynucleic acid from which it is derived.
The above-described nucleic acid fragment may be used as a specific hybridization probe for the detection of the nucleic acids of the current invention.
The term xe2x80x9cselectively hybridizingxe2x80x9d means that the hybridization signal obtained after hybridization of the fragment with the nucleic acid from which it is derived, is more intense than the hybridization signal obtained when the fragment is hybridized to the corresponding nucleic acid from another HIV-1 group O strain, under the same stringent hybridization and wash conditions. In practice this means that the nucleic acid fragment will show at least one mismatched nucleotide with the sequence of the corresponding nucleic acid fragment of another HIV-1 group O strain.
The term xe2x80x9cstringent hybridization conditionsxe2x80x9d implies that the hybridization takes place at a temperature which is situated approximately between Tm and (Tmxe2x88x9210xc2x0 C.), whereby Tm represents the calculated melting temperature of the target nucleic. It is generally known that the stringency depends on the percentage mismatches (=non-matching nucleotides upon alignment) present in the hybridizing duplex. According to a simplified formula, the hybridization temperature may be calculated as follows: Tmxe2x88x921.2 (% mismatch). A temperature decrease of 10xc2x0 C. implies a maximum percentage of allowed mismatches of 8.3%.
The invention further provides for a nucleic acid fragment consisting of a sequence of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of the sequence of a polynucleic acid as specified above, and characterized by the fact that it selectively amplifies the polynucleic acid from which it is derived.
The nucleic acid fragment as described above may be used as a specific amplification primer of the nucleic acids of the current invention.
The term xe2x80x9cselective amplificationxe2x80x9d refers to the fact that said nucleic acid fragment may initiate a specific amplification reaction of the nucleic acids of the invention (e.g. a polymerase chain reaction) in the presence of other nucleic acids, under appropriate amplification conditions. It means that, under the appropriate amplification conditions, only the nucleic acids of the invention will be amplified, and not the other nucleic acids possibly present.
Preferred embodiments of the invention comprise polynucleic acids or fragments thereof. as specified below.
A polynucleic acid comprising SEQ ID NO 1, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 1.
A polynucleic acid consisting of SEQ ID NO 1, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 1, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 1.
A polynucleic acid comprising SEQ ID NO 3, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 3.
A polynucleic acid consisting of SEQ ID NO 3, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 3, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 3.
A polynucleic acid comprising SEQ ID NO 5, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 5.
A polynucleic acid consisting of SEQ ID NO 5, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 5, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 5.
A polynucleic acid comprising SEQ ID NO 7, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 7.
A polynucleic acid consisting of SEQ ID NO 7, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 7, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 7.
A polynucleic acid comprising SEQ ID NO 9, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 9.
A polynucleic acid consisting of SEQ ID NO 9, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 9, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 9.
A polynucleic acid comprising SEQ ID NO 11, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 11.
A polynucleic acid consisting of SEQ ID NO 11, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to 50 contiguous nucleotides of SEQ ID NO 11, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 11.
A polynucleic acid comprising SEQ ID NO 13, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 13.
A polynucleic acid consisting of SEQ ID NO 13, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, up to the maximum number of contiguous nucleotides of SEQ ID NO 13, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 13.
A polynucleic acid comprising SEQ ID NO 15, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 15.
A polynucleic acid consisting of SEQ ID NO 15, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 15, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 15.
A polynucleic acid comprising SEQ ID NO 17, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 17.
A polynucleic acid consisting of SEQ ID NO 17, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 17, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 17.
A polynucleic acid comprising SEQ ID NO 19, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 19.
A polynucleic acid consisting of SEQ ID NO 19, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,50 up to the maximum number of contiguous nucleotides of SEQ ID NO 19, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 19.
A polynucleic acid comprising SEQ ID NO 21, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 21.
A polynucleic acid consisting of SEQ ID NO 21, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 21, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 21.
A polynucleic acid comprising SEQ ID NO 23, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 23.
A polynucleic acid consisting of SEQ ID NO 23, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 23, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 23.
A polynucleic acid comprising SEQ ID NO 25, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 25.
A polynucleic acid consisting of SEQ ID NO 25, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to 50 contiguous nucleotides of SEQ ID NO 25, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 25.
A polynucleic acid comprising SEQ ID NO 27, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 27.
A polynucleic acid consisting of SEQ ID NO 27, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 27, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 27.
A polynucleic acid comprising SEQ ID NO 29, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 29.
A polynucleic acid consisting of SEQ ID NO 29, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 29, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 29.
A polynucleic acid comprising SEQ ID NO 31, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 31.
A polynucleic acid consisting of SEQ ID NO 31, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 31, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 31.
A polynucleic acid comprising SEQ ID NO 33, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 33.
A polynucleic acid consisting of SEQ ID NO 33, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 33, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 33.
A polynucleic acid comprising SEQ ID NO 35, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 35.
A polynucleic acid consisting of SEQ ID NO 35, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 35, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 35.
A polynucleic acid comprising SEQ ID NO 37, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 37.
A polynucleic acid consisting of SEQ ID NO 37, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 37, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 37.
A polynucleic acid comprising SEQ ID NO 39, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 39.
A polynucleic acid consisting of SEQ ID NO 39, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,50 up to the maximum number of contiguous nucleotides of SEQ ID NO 39, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 39.
A polynucleic acid comprising SEQ ID NO 41, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 41.
A polynucleic acid consisting of SEQ ID NO 41, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 41, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 41.
A polynucleic acid comprising SEQ ID NO 43, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 43.
A polynucleic acid consisting of SEQ ID NO 43, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 43, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 43.
A polynucleic acid comprising SEQ ID NO 45, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 45.
A polynucleic acid consisting of SEQ ID NO 45, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 45, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 45.
A polynucleic acid comprising SEQ ID NO 47, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 47.
A polynucleic acid consisting of SEQ ID NO 47, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 47, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 47.
A polynucleic acid comprising SEQ ID NO 49, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 49.
A polynucleic acid consisting of SEQ ID NO 49, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 49, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 49.
A polynucleic acid comprising SEQ ID NO 51, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 51.
A polynucleic acid consisting of SEQ ID NO 51, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 51, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 51.
A polynucleic acid comprising SEQ ID NO 106, or a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 106.
A polynucleic acid consisting of SEQ ID NO 106, or a fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous nucleotides of SEQ ID NO 106, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid consisting of SEQ ID NO 106.
The invention further provides for a virus strain belonging to HIV-1 group O, comprising in its genome any of the above-mentioned nucleic acids.
More particularly, the invention provides for a virus strain belonging to HIV-1 group O, comprising in its genome the RNA equivalent of
one of the DNA sequences represented by SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 106 and/or
one of the DNA sequences represented by SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, and/or
a variant sequence of the above-mentioned DNA sequences, said variant sequence showing at least 95% homology with the entire length of one of the above-mentioned sequences.
More particularly, the invention relates to a strain of HIV-1 group O as defined above, comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 5 and/or SEQ ID NO 43 or a variant sequence thereof, said variant sequence showing at least 95% homology with SEQ ID NO 5 and/or SEQ ID NO 43. An HIV-1 group O strain of this type, termed FABA (or synonymously MP331) has been deposited at the ECACC on Jun. 13 1997, under accession No V97061301.
An example of a variant sequence of SEQ ID NO 5 is SEQ ID NO 7. The latter sequence was determined on a serum sample of a patient infected by the strain FABA, while the former sequence was determined on peripheral blood mononuclear cells (PBMC""s) taken from the same patient. The nucleic acids represented by SEQ ID NO 5 and SEQ ID NO 7 show 95% homology, and can be said to belong to strains from the same quasi-species.
The invention also relates to a strain of HIV-1 group O as defined above, comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 9 and/or SEQ ID NO 45 or a variant sequence, said variant sequence showing at least 95% homology with SEQ ID NO 9 and/or SEQ ID NO 45. An HIV-1 group O strain of this type, termed MP450, has been deposited at the ECACC on Jun. 13, 1997 under accession No. V97061302.
An example of a variant sequence of SEQ ID NO 9 is SEQ ID NO 11. The latter sequence was determined on a serum sample of a patient infected by the strain MP450, while the former sequence was determined on peripheral blood mononuclear cells (PBMC""s) taken from the same patient.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above, comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 39 and/or SEQ ID NO 51 or a variant sequence, said variant sequence showing at least 95% homology with SEQ ID NO 39 and/or SEQ ID NO 51. An HIV-1 group O strain of this type, termed MP539, has been deposited at the ECACC on Jun. 13, 1997 under accession No. V97061303.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 1 and/or SEQ ID NO 41 or a variant sequence, said variant sequence showing at least 95% homology with SEQ ID NO 1 and/or SEQ ID NO 41. A strain of this type is termed MP340 throughout this invention.
An example of a variant sequence of SEQ ID NO 1 is SEQ ID NO 3. The latter sequence was determined on a serum sample of a patient infected by the strain MP340, while the former sequence was determined on peripheral blood mononuclear cells (PBMC""s) taken from the same patient. The nucleic acids represented by SEQ ID NO 1 and SEQ ID NO 3 show 99% homology, and can be said to belong to strains from the same quasi-species.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 13 and/or SEQ ID NO 47 or a variant sequence, said variant sequence showing at least 95% homology with SEQ ID NO 13 and/or SEQ ID NO 47. A strain of this type is termed MP448 throughout this invention.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 17 and/or SEQ ID NO 49 or a variant sequence, said variant sequence showing at least 95% homology with SEQ ID NO 17 and/or SEQ ID NO 49. A strain of this type is termed 189 throughout this invention.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 19 or a variant sequence showing at least 95% homology with SEQ ID NO 19. A strain of this type is termed 320 throughout this invention.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 21 or a variant sequence showing at least 95% homology with SEQ ID NO 21. A strain of this type is termed BSD422 throughout this invention.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 23 or a variant sequence showing at least 95% homology with SEQ ID NO 23. A strain of this type is termed KGT008 throughout this invention.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 25 or a variant sequence showing at least 95% homology with SEQ ID NO 25. A strain of this type, termed MP575, has been deposited at the ECACC on Jul. 13, 1998, under provisional accession No. V98071301.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 27 or a variant sequence showing at least 95% homology with SEQ ID NO 27. A strain of this type is termed BSD189 throughout this invention.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 29 or a variant sequence showing at least 95% homology with SEQ ID NO 29. A strain of this type is termed BSD649 throughout this invention.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 31 or a variant sequence showing at least 95% homology with SEQ ID NO 31. A strain of this type is termed BSD242 throughout this invention.
Furthermore, the invention also relates to a strain of HIV-1 group-O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 33 or a variant sequence showing at least 95% homology with SEQ ID NO 33. A strain of this type is termed 533 throughout this invention.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 35 or a variant sequence showing at least 95% homology with SEQ ID NO 35. A strain of this type is termed 772. P94 throughout this invention.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 37 or a variant sequence showing at least 95% homology with SEQ ID NO 37. A strain of this type is termed MP95B throughout this invention.
Furthermore, the invention also relates to a strain of HIV-1 group O as defined above comprising in its genome the RNA equivalent of the DNA sequence represented by SEQ ID NO 106 or a variant sequence showing at least 95% homology with SEQ ID NO 106. A strain of this type, termed MP645, has been deposited at the ECACC on Jul. 13, 1998, under provisional accession No. V98071302.
Another embodiment of the current invention provides for a nucleic acid molecule isolated from any of the HIV-1 group O strains as defined above.
In addition, the current invention provides for an antigen or antigen fragment isolated from any of the HIV-1 group O strains as defined above.
It is to be understood that the current invention also provides for nucleic acid sequences and antigen sequences which are contained in the above-mentioned new HIV-1 group O viral strains, and which extend beyond the explicitly cited sequences represented by SEQ ID NO 1 to 102, 106, 135 to 138. The person skilled in the art will realize that, starting from the partial sequences disclosed above, it is perfectly possible to obtain the complete genomic information of the respective viruses, by standard cloning methods such as the construction of a cDNA library or the construction of a genomic library or by the technique of the polymerase chain reaction. Sometimes a combination of these methods may be necessary to obtain the sequence of the full genome.
The following describes the strategies which may be followed to obtain additional genomic sequence information on HIV-1 group O strains, of which partial sequences have been disclosed above.
HIV-1 group O viruses are propagated and isolated using standard methods e.g. by cultivation of peripheral blood lymphocytes (PBMC) from the HIV-infected individual together with stimulated lymphocytes from healthy donors, or alternatively by infecting cell lines with the virus in a permanent way. Once virus is detected in the culture supernatant using standard techniques (e.g. measuring reverse transcriptase activity; measuring p24 antigen . . . ), virus is harvested from the culture supernantant by centrifugation under conditions where the virus is pelleted. RNA is obtained by disrupting the virus in a buffer containing 6M guanidinium chloride and the RNA is pelleted through a 5.5M CsCl cushion. The RNA which is resuspended in a suitable buffer is then phenolized and precipitated with e.g. ethanol and lithium chloride.
cDNA synthesis is performed on the complete RNA or part of the RNA using commercially available kits. OligodT primers, random primers, or HIV-1 specific primers may be used to prime the cDNA synthesis which is done by a reverse transcriptase (RT) enzyme. This leads to a first DNA strand which is complementary to the initial RNA strand and which forms RNA::DNA hybrids. The RNA strand is removed with Rnase H and the second DNA strand is then synthesised with DNA polymerase I. The overhanging single stranded cDNA ends are removed with T4 DNA polymerase. The resulting cDNA is ligated to linkers which contain an appropriate restriction site. After hydrolysis of the cDNA with the appropriate restriction enzyme, the cDNA of suitable size is isolated (e.g. from agarose gel after electrophoresis) and ligated in a suitable vector. The vector containing the cDNA fragments can be propagated in competent E. coli cells using standard methods.
Various techniques to screen for colonies containing HIV-1 specific sequences are known in the art. They involve screening of e.g. a cDNA expression library (e.g. xcexgt11) with serum (polyclonal or monoclonal serum) or the screening of a cDNA library with 32P labelled HIV-1 DNA fragments under non-stringent or stringent hybridization conditions. Background signals are lowered by washing the filters subsequently under more stringent conditions. After identification of the E. coli containing the suitable fragment, the fragment is isolated from the plasmid and is introduced (as a complete entity or a fragment thereof) in expression vectors. Using standard techniques, these vectors produce the protein(s) encoded by the inserted DNA fragment. The resulting proteins is further purified and used for the development of diagnostic assays. Sequence information of the virus is obtained from the plasmid containing viral DNA sequences.
Chromosomal DNA is prepared from cells infected with the HIV-1 group O virus (e.g. cells permanently producing the virus) using standard techniques (Maniatis et al. 1982). This DNA may be used to construct a genomic library (Zabarousky and Allikmets 1986). The chromosomal DNA which contains the proviral HIV-1 group O DNA is partially digested with a selected restriction enzyme. Fragments between 9 Kb and 23 Kb, isolated on a 40%-10% sucrose gradient, are manipulated according to standard techniques in order to introduce them in a vector system suitable for the cloning of long DNA fragments e.g. lambda derived vectors or cosmids.
The vector with the DNA fragment is introduced in a suitable E. coli strain and is further propagated onto plates. Plaques or colonies from the genomic library are transferred to nylon or nitrocellulose membranes and screened with enzyme or 32P labelled DNA fragments of the viral genome (plaque or colony screening) under non-stringent or stringent hybridization conditions. Colonies or plaques displaying positive signals are purified from other colonies or plaques. The viral DNA is further subcloned and sequenced. Genes or fragments of genes are further manipulated using standard techniques in order to express important viral proteins or epitopes which may be used for the development of diagnostic assays.
HIV-1 group O viral DNA fragments may also be obtained using the polymerase chain reaction (PCR) (Kwok et al. 1987) which is a standard technology used for the cloning of DNA fragments. PCR may be performed on cellular DNA of cells infected with the virus or on cDNA obtained from viral RNA derived from virus culture, lymphocytes, serum, plasma, . . . . The PCR may use primers which contain specific sequences of the virus based on sequences of the virus which are already known, or alternatively, primers which contain sequences derived from related viruses in regions known to be conserved or not conserved among HIV variants. Annealing conditions of the primers should preferentially not be too stringent (e.g. Tmxe2x88x9220xc2x0 C.), however the best conditions should be experimentally established. The resulting amplification product is subsequently sequenced and new primers are designed based on the newly generated sequence in order to further amplify the viral DNA, again eventually in combination with primers derived from the partially determined sequence of the isolate or from the sequence of related viruses.
Example 4 provides the cloning and sequencing strategy followed in order to obtain the polynucleic acid sequences encoding the antigens Vif, Pol, Vpr, Tat (1st exon), Vpu, Rev (1st exon) and gp160, or fragments thereof, from the HIV-1 group O viruses FABA (MP331), MP448, MP539 and MP 645.
Therefore, in addition to the gp41 and V3 sequences described above, the present invention further provides for a polynucleic acid containing a polynucleic acid sequence encoding at least part of the Vif, Pol, Vpr, Tat (1st exon), Vpu, Rev (1st exon) or gp160 antigens from any of the following HIV-1 group O strains of the invention: MP340, FABA(MP331), MP450, MP448, 189, MP539, 320, BSD422, KGT008, MP575, BSD189, BSD649, BSD242, 533, 772P94, MP95B and MP645.
The terms xe2x80x9cVif, Pol, Vpr, Tat (1st exon), Vpu, Rev (1st exon) and gp160xe2x80x9d are terms for HIV-antigens familiar to the person skilled in the art. Vif and Vpu have important roles during virion morphogenesis. Vif is required for the production of fully infectious viruses, while Vpu is necessary for the efficient release of virus particles budding from the cell membrane in cultured cells (Gxc3x6ttlinger et al, 1993). Vpu also mediates the rapid degradation of the CD4 receptor molecule in the endoplasmatic reticulum (Willey et al, 1992, Bour et al, 1995). The Vpr protein is involved in the nuclear migration of the prointegration complex (Heinzinger et al, 1994) and is also found in mature virions and hence a structural component of the virus (Paxton et al, 1993). Tat (transactivator) and Rev (regulator of virion expression) are encoded in overlapping reading frames which generate small regulatory proteins translated from multiple spliced mRNAs (Salfeld et al, 1990; Solomin et al, 1990, Furtado et al, 1991). Both proteins are essential for virus replication and are positive regulators of gene expression (Arya et al, 1985; Feinberg et al, 1986). Pol is encoded by the coding gene pol, which overlaps with the gag information but in a different reading frame. Pol is a precursor protein which is autocleaved to form the following viral enzymes: a protease, a reverse transcriptase with polymerase activity and Rnase H activity, and an integrase (Ross et al, 1991). FIG. 8 illustrates the sequence of a large genomic fragment from a number of HIV-1 group O strains of the current invention (MP645 (SEQ ID NO 106), MP331 (SEQ ID NO 103), MP448 (SEQ ID NO 104) and MP539(SEQ ID NO 105)), and the location of the Vif, Pol, Vpr, Tat (1st exon), Vpu, Rev (1st exon) and gp160 (partially) genes in these sequences.
The present invention thus provides for a polynucleic acid comprising a nucleotide sequence chosen from the group of
(I) a nucleotide sequence represented by any of SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, or
(ii) a nucleotide sequence complementary to a sequence according to (I), or
(iii) a nucleotide sequence showing at least 95%, preferably 96%, 97%, 98% and most preferably 99% homology to a sequence according to (I), or
(iv) a nucleotide sequence according to (I) whereby T is replaced by U, or
(v) a nucleotide sequence according to (I) whereby at least one nucleotide is substituted by a nucleotide analogue, or
(vi) a fragment a sequence of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of any of the nucleotide sequences according to (I) to (v), and characterized by the fact that it selectively hybridizes to the polynucleic acid from which it is derived, and/or selectively amplifies the polynucleic acid from which it is derived.
More particularly, the present invention provides for a polynucleic acid consisting of a nucleotide sequence chosen from the group of
(I) a nucleotide sequence represented by any of SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, or
(ii) a nucleotide sequence complementary to a sequence according to (I), or
(iii) a nucleotide sequence showing at least 95%, preferably 96%, 97%, 98% and most preferably 99% homology to a sequence according to (I), or
(iv) a nucleotide sequence according to (I) whereby T is replaced by U, or
(v) a nucleotide sequence according to (I) whereby at least one nucleotide is substituted by a nucleotide analogue
(vi) a fragment a sequence of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of any of the nucleotide sequences according to (I) to (v), and characterized by the fact that it selectively hybridizes to the polynucleic acid from which it is derived, and/or selectively amplifies the polynucleic acid from which it is derived.
The above-described nucleic acid fragment may be used as a specific hybridization probe for the detection of the nucleic acids of the current invention.
The term xe2x80x9cselectively hybridizingxe2x80x9d means that the hybridization signal obtained after hybridization of the fragment with the nucleic acid from which it is derived, is more intense than the hybridization signal obtained when the fragment is hybridized to the corresponding nucleic acid from another HIV-1 group O strain, under the same stringent hybridization and wash conditions. In practice this means that the nucleic acid fragment will show at least one mismatched nucleotide with the sequence of the corresponding nucleic acid fragment of another HIV-1 group O strain.
The term xe2x80x9cstringent hybridization conditionsxe2x80x9d implies that the hybridization takes place at a temperature which is situated approximately between Tm and (Tmxe2x88x9210xc2x0 C.), whereby Tm represents the calculated melting temperature of the target nucleic. It is generally known that the stringency depends on the percentage mismatches (=non-matching nucleotides upon alignment) present in the hybridizing duplex. According to a simplified formula, the hybridization temperature may be calculated as follows: Tmxe2x88x921.2 (% mismatch). A temperature decrease of 10xc2x0 C. implies a maximum percentage of allowed mismatches of 8.3%.
The nucleic acid fragment as described above may also be used as a specific amplification primer of the nucleic acids of the current invention.
The term xe2x80x9cselective amplificationxe2x80x9d refers to the fact that said nucleic acid fragment may initiate a specific amplification reaction of the nucleic acids of the invention (e.g. a polymerase chain reaction) in the presence of other nucleic acids, under appropriate amplification conditions. It means that, under the appropriate amplification conditions, only the nucleic acids of the invention will be amplified, and not the other nucleic acids possibly present.
Preferred embodiments of the invention comprise polynucleic acids or fragments thereof. as specified below.
A polynucleic acid comprising SEQ ID NO 103, or comprising a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 103.
A polynucleic acid comprising SEQ ID NO 104, or comprising a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 104.
A polynucleic acid comprising SEQ ID NO 105, or comprising a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 105.
A polynucleic acid comprising SEQ ID NO 106, or comprising a fragment consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to 50 contiguous nucleotides of said polynucleic acid, said fragment characterized by the fact that it selectively hybridizes to the polynucleic acid comprising SEQ ID NO 106.
A polynucleic acid fragment consisting of a sequence of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to 50 contiguous nucleotides of SEQ ID NO 103, said fragment characterized by the fact that it selectively amplifies the polynucleic acid from which it is derived.
A polynucleic acid fragment consisting of a sequence of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to 50 contiguous nucleotides of SEQ ID NO 104, said fragment characterized by the fact that it selectively amplifies the polynucleic acid from which it is derived.
A polynucleic acid fragment consisting of a sequence of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to 50 contiguous nucleotides of SEQ ID NO 105, said fragment characterized by the fact that it selectively amplifies the polynucleic acid from which it is derived.
A polynucleic acid fragment consisting of a sequence of at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to 50 contiguous nucleotides of SEQ ID NO 106, said fragment characterized by the fact that it selectively amplifies the polynucleic acid from which it is derived.
The invention further provides for an antigen comprising at least part of Vif, Pol, Vpr, Tat (1st exon), Vpu, Rev (1st exon) and/or gp160 encoded by the nucleic acid sequences as described above from any of the following HIV-1 group O strains: MP340, FABA(MP331), MP450, MP448, 189, MP539, 320, BSD422, KGT008, MP575, BSD189, BSD649, BSD242, 533, 772P94, MP95B and MP645.
The current invention more particularly provides for an antigen comprising at least one amino acid sequence chosen from the following groups of sequences
Ii) an amino acid sequence represented by any of SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109 and SEQ ID NO 110 representing the Vif antigen,
(ii) an amino acid sequence represented by any of SEQ ID NO 111, SEQ ID NO 112, SEQ ID NO 113 and SEQ ID NO 114 representing the Vpu antigen,
(iii) an amino acid sequence represented by any of SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117 and SEQ ID NO 118 representing the Vpr antigen,
(iv) an amino acid sequence represented by any of SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121 and SEQ ID NO 122 representing the Tat antigen,
(v) an amino acid sequence represented by any of SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125 and SEQ ID NO 126 representing the Rev antigen,
(vi) an amino acid sequence represented by any of SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129 and SEQ ID NO 130 representing the Pol antigen,
(vii) an amino acid sequence represented by any of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 2, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 132, and SEQ ID NO 134 representing at least part of the Env antigen, or
(viii) a fragment of any of the above-mentioned antigens Ii) to (vii), said fragment consisting of at least 8, preferably 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous amino acids of the amino acid sequence of said antigen, and being characterized by the fact that it specifically reacts with antibodies raised against said antigen.
The current invention further provides for an antigen consisting of an amino acid sequence chosen from the following groups of sequences:
Ii) an amino acid sequence SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109 and SEQ ID NO 110 representing the Vif antigen,
(i) an amino acid sequence SEQ ID NO 111, SEQ ID NO 112, SEQ ID NO 113 and SEQ ID NO 114 representing the Vpu antigen,
(iii) an amino acid sequence SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117 and SEQ ID NO 118 representing the Vpr antigen,
(iv) an amino acid sequence SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121 and SEQ ID NO 122 representing the Tat antigen,
(v) an amino acid sequence SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125 and SEQ ID NO 126 representing the Rev antigen,
(vi) an amino acid sequence SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129 and SEQ ID NO 130 representing the Pol antigen,
(vii) an amino acid sequence represented by any of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 132, and SEQ ID NO 134 representing at least part of the Env antigen, or
(viii) a fragment of any of the above-mentioned antigens (I) to (vii), said fragment consisting of at least 8, preferably 9, 10,11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 50 up to the maximum number of contiguous amino acids of the amino acid sequence of said antigen, and being characterized by the fact that it specifically reacts with antibodies raised against said antigen.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain MP340 comprising at least one of the sequences according to SEQ ID NO 2, 4, 42, 59, 60, 61, 73, 85, 86, 100, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) a polynucleic acid encoding an antigen according to (I) and comprising at least one of the nucleotide sequences according to SEQ ID NO 1, 3, 41, including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iii) a virus strain comprising in its genome a polynucleic acid according to (ii), more particularly a virus strain termed MP340, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain MP331 (or FABA) comprising at least one of the sequences according to SEQ ID NO 6, 8, 44, 56, 57, 58, 82, 83, 84, 138 or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) an antigen derived from the Vif, Vpu, Vpr, Tat, Rev, Pol and Env protein of the new HIV-1 group O strain MP331 (or FABA) comprising at least one of the sequences according to SEQ ID NO 107, 111, 115, 119, 123, 127, 131, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(iii) a polynucleic acid encoding an antigen according to (I) or (ii) and comprising at least one of the nucleic acid sequences according to SEQ ID NO 5, 7, 43, 103, including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iv) a virus strain comprising in its genome said a polynucleic acid according to (iii), more particularly a virus strain termed MP331 (FABA) deposited at the ECACC under accession number V97061301, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain MP450 comprising at least one of the sequences according to SEQ ID NO 10, 12, 46, 62, 63, 64, 73, 87, 100, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) a polynucleic acid encoding an antigen according to (I) and comprising at least one of the nucleotide sequences according to SEQ ID NO 9, 11, 45, including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iii) a virus strain comprising in its genome a polynucleic acid according to (ii), more particularly a virus strain termed MP450 deposited at the ECACC under accession number V97061302, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain MP448 comprising at least one of the sequences according to SEQ ID NO 14, 16, 48, 65, 66, 67, 76, 88, 101, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) an antigen derived from the Vif, Vpu, Vpr, Tat, Rev, Pol and Env protein of the new HIV-1 group O strain MP448 comprising at least one of the sequences according to SEQ ID NO 108, 112, 116, 120, 124, 128, 132 or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(iii) a polynucleic acid encoding an antigen according to (I) or (ii) and comprising at least one of the nucleic acid sequences according to SEQ ID NO 13, 15, 47, 104, including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iv) A virus strain comprising in its genome said polynucleic acid according to (iii), more particularly a virus strain termed MP448, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain 189 comprising at least one of the sequences according to SEQ ID NO 18, 50, 53, 54, 55, 73, 90, 91, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) a polynucleic acid encoding an antigen according to (I) and comprising at least one of the nucleotide sequences according to SEQ ID NO 17, 49, including homologous sequences, complementary sequences, and fragments hybridizing thereto,
(iii) a virus strain comprising in its genome a polynucleic acid according to (ii), more particularly a virus strain termed 189, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain MP539 comprising at least one of the sequences according to SEQ ID NO 40, 52, 68, 69, 70, 71, 89, 95 or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) an antigen derived from the Vif, Vpu, Vpr, Tat, Rev, Pol and Env protein of the new HIV-1 group O strain MP539 comprising at least one of the sequences according to SEQ ID NO 109, 113, 117, 121, 125, 129, 133, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from.
(iii) a polynucleic acid encoding an antigen according to (I) or (ii) and comprising at least one of the nucleic acid sequences according to SEQ ID NO 39, 51, 105, including homologous sequences, complementary sequences, and fragments hybridizing thereto,
(iv) A virus strain comprising in its genome said polynucleic acid according to (iii), more particularly a virus strain deposited at the ECACC under accession number V97061303, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain 320 comprising at least one of the sequences according to SEQ ID NO 20, 92, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) a polynucleic acid encoding an antigen according to (I) and comprising the nucleotide sequence according to SEQ ID NO 19 including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iii) a virus strain comprising in its genome a polynucleic acid according to (ii), more particularly a virus strain termed 320, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain BSD422 comprising at least one of the sequences according to SEQ ID NO 22, 80, 79, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) a polynucleic acid encoding an antigen according to (I) and comprising the nucleotide sequence according to SEQ ID NO 21 including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iii) a virus strain comprising in its genome a polynucleic acid according to (ii), more particularly a virus strain termed BSD422, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain KGT008 comprising at least one of the sequences according to SEQ ID NO 24, 79, 99? or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) a polynucleic acid encoding an antigen according to (I) and comprising the nucleotide sequence according to SEQ ID NO 23 including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iii) a virus strain comprising in its genome a polynucleic acid according to (ii), more particularly a virus strain termed KGT008, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain MP575 comprising the sequence according to SEQ ID NO 26, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) a polynucleic acid encoding an antigen according to Ii) and comprising the nucleotide sequence according to SEQ ID NO 25 including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iii) A virus strain comprising in its genome said polynucleic acid according to (ii), more particularly a virus strain deposited at the ECACC under provisional accession number V98071301, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain BSD189 comprising at least one of the sequences according to SEQ ID NO 28, 72, 95, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) a polynucleic acid encoding an antigen according to (I) and comprising the nucleotide sequence according to SEQ ID NO 27 including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iii) a virus strain comprising in its genome a polynucleic acid according to (ii), more particularly a virus strain termed BSD189, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain BSD649 comprising at least one of the sequences according to SEQ ID NO 30, 77, 98, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) a polynucleic acid encoding an antigen according to (I) and comprising the nucleotide sequence according to SEQ ID NO 29 including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iii) a virus strain comprising in its genome a polynucleic acid according to (ii), more particularly a virus strain termed BSD649, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain BSD242 comprising at least one of the sequences according to SEQ ID NO 32, 81, 96, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) a polynucleic acid encoding an antigen according to (I) and comprising the nucleotide sequence according to SEQ ID NO 31 including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iii) a virus strain comprising in its genome a polynucleic acid according to (ii), more particularly a virus strain termed BSD242, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain 533 comprising at least one of the sequences according to SEQ ID NO 34, 81, 93, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) a polynucleic acid encoding an antigen according to (I) and comprising the nucleotide sequence according to SEQ ID NO 33 including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iii) a virus strain comprising in its genome a polynucleic acid according to (ii), more particularly a virus strain termed 533, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain 772P94 comprising at least one of the sequences according to SEQ ID NO 36, 75, 94, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) a polynucleic acid encoding an antigen according to (I) and comprising the nucleotide sequence according to SEQ ID NO 35 including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iii) a virus strain comprising in its genome a polynucleic acid according to (ii), more particularly a virus strain termed 772P94, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O strain MP95B comprising at least one of the sequences according to SEQ ID NO 38, 74, 102, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) a polynucleic acid encoding an antigen according to (I) and comprising the nucleotide sequence according to SEQ ID NO 37 including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iii) a virus strain comprising in its genome a polynucleic acid according to (ii), more particularly a virus strain termed MP95B, as well as polynucleic acids and antigens isolated therefrom.
The current invention thus also relates to:
(I) an antigen derived from the gp160 env precursor protein of the new HIV-1 group O stain MP645 comprising at least one of the sequences according to SEQ ID NO 135, 136, 137, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from;
(ii) an antigen derived from the Vif, Vpu, Vpr, Tat, Rev, Pol and Env protein of the new HIV-1 group O strain MP645 comprising one of the sequences according to SEQ ID NO 110, 114, 118, 122, 126, 130, 134, or fragments thereof with said fragments specifically reacting with antibodies raised against the antigen they are derived from.
(iii) a polynucleic acid encoding an antigen according to (I) or (ii) and comprising the nucleic acid sequence according to SEQ ID NO 106, including homologous sequences, complementary sequences, and fragments hybridizing thereto;
(iv) A virus strain comprising in its genome said polynucleic acid according to (iii), more particularly a virus strain deposited at the ECACC under provisional accession number V98071302, as well as polynucleic acids and antigens isolated therefrom.
In another embodiment, the invention provides for an antibody, preferably a monoclonal antibody, raised against an antigen or antigen fragment as described above. Such an antibody recognizes specifically the antigen or the antigen fragment to which it has been raised.
According to an alternative embodiment, the present invention also relates to an antigen-binding fragment of the antibody, said fragment being of the F(abxe2x80x2)2, Fab or single chain Fv type, or any type of recombinant antibody derived from said specific antibodies or monoclonal antibodies, provided that said antibody fragment or recombinant antibody still recognizes specifically the antigen or antigen fragment to which it has been raised.
The expression xe2x80x9cantibody recognizing specificallyxe2x80x9d means that the binding between the antigen as a ligand and a molecule containing an antibody combining site, such as a Fab portion of a whole antibody, is specific, signifying that no cross-reaction occurs.
The expression xe2x80x9cantibody specifically raised against a compoundxe2x80x9d means that the sole immunogen used to produce said antibody was said compound.
The possible cross-reactivity of polyclonal antisera may be eliminated by preabsorption of the polyclonal antiserum against the cross-reacting antigenic determinants.
In a preferential embodiment, the above-mentioned antibodies are neutralizing antibodies, i.e. antibodies capable of in vitro inhibition of viral growth, determined according to methods known in the art.
Neutralizing antibodies may be used as a reagent in a so-called xe2x80x9cpassive vaccinexe2x80x9d composition, i.e. a composition conferring temporary protection against an infection, upon injection in an individual. The invention also relates to passive vaccine compositions, comprising any of the above-mentioned neutralizing antibodies.
The monoclonal antibodies of the invention can be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat, immunized with the antigen of the invention, defined above on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the antigen which has been initially used for the immunization of the animals.
The monoclonal antibodies according to a preferred embodiment of the invention may be humanized versions of the mouse monoclonal antibodies made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains.
Also fragments derived from these monoclonal antibodies such as Fab, F(ab)xe2x80x22 and ssFv (xe2x80x9csingle chain variable fragmentxe2x80x9d), providing they have retained the original binding properties, form part of the present invention. Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases. It is well known to the person skilled in the art that monoclonal antibodies, or fragments thereof, can be modified for various uses.
The antibodies involved in the invention can be labelled by an appropriate label of the enzymatic, fluorescent, or radioactive type.
The invention also relates to the use of the antigens of the invention, or fragments thereof, for the selection of recombinant antibodies by the process of repertoire cloning (Perrson et al., 1991).
The present invention further relates to an anti-idiotype antibody raised against any of the antibodies as defined above.
The term xe2x80x9canti-idiotype antibodiesxe2x80x9d refers to monoclonal antibodies raised against the antigenic determinants of the variable region of monoclonal antibodies themselves raised against the antigens of the invention. These antigenic determinants of immunoglobulins are known as idiotypes (sets of idiotopes) and can therefore be considered to be the xe2x80x9cfingerprintxe2x80x9d of an antibody (for review see de Prxc3xa9val, 1978; Fleishmann and Davie,1984). The methods for production of monoclonal anti-idiotypic antibodies have been described by Gheuens and McFarlin (1982). Monoclonal anti-idiotypic antibodies have the property of forming an immunological complex with the idiotype of the monoclonal antibody against which they were raised. In this respect the monoclonal antibody is often referred to as Ab1, and the anti-idiotypic antibody is referred to as Ab2. These anti-idiotype Ab2s may be used as substitutes for the polypeptides of the invention or as competitors for binding of the polypeptides of the invention to their target.
The present invention further relates to antisense peptides derived from the antigens of the invention as described above.
More particularly, the term xe2x80x9cantisense peptidexe2x80x9d is reviewed by Blalock (1990) and by Roubos (1990). In this respect, the molecular recognition theory (Blalock, 1990) states that not only the complementary nucleic acid sequences interact but that, in addition, interacting sites in proteins are composed of complementary amino acid sequences (sense ligand with receptor or sense ligand with antisense peptides). Thus, two peptides derived from complementary nucleic acid sequences in the same reading frame will show a total interchange of their hydrophobic and hydrophilic amino acids when the amino terminus of one is aligned with the carboxy terminus of the other. This inverted hydropathic pattern might allow two such peptides to assume complementary conformations responsible for specific interaction.
The antisense peptides can be prepared as described in Ghiso et al. (1990). By means of this technology it is possible to logically construct a peptide having a physiologically relevant interaction with a known peptide by simple nucleotide sequence analysis for complementarity, and synthesize the peptide complementary to the binding site.
The present invention further relates to a diagnostic method for detecting the presence of an HIV-1 infection, said method comprising
the detection of antibodies against HIV-1, including HIV-group O, using any of the antigens or antigen fragments of the invention as described above, and/or
the detection of viral antigen originating from HIV-1, including HIV-1 group O, using any of the antibodies of the invention as described above and/or
the detection of viral nucleic acids originating from HIV-1, including HIV-1 group O, using any of the nucleic acids or nucleic acid fragments of the invention as described above, in a biological sample.
Preferably the above-mentioned diagnostic method for detecting the presence of an HIV-1 infection also includes the detection of an HIV-1 group O infection, and more particularly also includes the detection of an infection caused by any of the HIV-1 group O strains of the current invention.
The term xe2x80x9cbiological samplexe2x80x9d refers to any biological sample (tissue or fluid) possibly containing HIV nucleic acids, and/or HIV antigens and/or antibodies against HIV, and refers more particularly to blood, serum, plasma, organs or tissue samples.
In most instances, the [HIV-1 group O]-reagents (=antigens and/or antibodies and/or nucleic acids) of the invention will be used in methods which combine them with other HIV-reagents (=antigens and/or antibodies and/or nucleic acids). The addition of the HIV-1 type O reagents of the current invention to methods and kits for detection of HIV-infection in general, may result in methods and kits showing
a higher sensitivity, and/or
a higher discriminating power between different types of HIV-infection, for example HIV-1 group M, HIV-1 group O and HIV-2 infection.
The term xe2x80x9csensitivityxe2x80x9d refers to the ratio of positively reacting samples/the number of truly infected samples.
More specifically, the present invention relates to a method for in vitro diagnosis of a HIV-1 infection, including a HIV-1 group O infection, comprising at least the step of contacting a biological sample with:
a HIV-1 group O antigen, or antigen fragment, as defined above, under conditions allowing the formation of an immunological complex, and/or,
a HIV-1 group O nucleic acid, or nucleic acid fragment, as defined above, under conditions allowing the formation of a hybridization complex, with the nucleic acids of said sample being possibly amplified prior to hybridization, and/or,
an antibody specifically directed against an HIV-1 group O antigen as defined above, under conditions allowing the formation of an immunological complex, and/or,
an anti-idiotype antibody as defined above, under conditions allowing the formation of an antibody-anti-idiotypic complex, and/or,
an antisense peptide as defined above, under conditions allowing the formation of an antigen-antisense peptide complex,
and subsequently detecting the complexes formed.
In a more specific embodiment, the invention relates to a method for detecting the presence of antibodies against HIV-1 in a biological sample, in particular antibodies against an HIV-1 group O strain, preferably a serum sample, comprising the following steps:
contacting the biological sample taken from a patient with at least one antigen or antigen fragment as described above, under conditions enabling the formation of an immunological complex, and
detecting the immunological complex formed between said antigen or antigen fragment and the antibodies possibly present in the sample.
Conditions allowing the formation of an immunological complex are known to the person skilled in the art.
In a special embodiment, the antigens being used in the above-described method for detection of anti-HIV-1 group O antibodies, can be replaced by anti-idiotype antibodies as described above, acting as their equivalents.
Conditions allowing the formation of an antibody-anti-idiotypic complex are known in the art.
The invention farther relates to a method for detecting the presence of an antigen or an antigen fragment of HIV-1, in particular an antigen or antigen fragment of an HIV-1 group O strain, in a biological sample comprising the following steps:
contacting the biological sample taken from a patient with at least one antibody as described above under conditions enabling the formation of an immunological complex, and
detecting the immunological complex formed between said antibody and the antigen or antigen fragment possibly present in the sample.
In a special embodiment, the antibodies being used in the above-described method for detection of HIV-1 group O antigens, may be replaced by anti-sense peptides as described above, acting as their equivalents.
Conditions allowing the formation of an antigen-antisense peptide complex are known in the art.
Design of immunoassays is subject to a great deal of variation, and many formats are known in the art. Protocols may, for example, use solid supports, or immunoprecipitation. Most assays involve the use of labelled antibody or polypeptide; the labels may be, for example, enzymatic, fluorescent, chemoluminescent, radioactive, or dye molecules. Assays which amplify the signals from the immune complex are also known, examples of which are assays which utilize biotin and avidin or streptavidin, and enzyme-labelled and mediated immunoassays, such as ELISA assays.
An advantageous embodiment provides for a method for detection of anti-HIV-1 group O antibodies in a sample, whereby the antigens or antigen fragments of the invention are immobilized on a solid support, for example on a membrane strip. Different antigens or antigen fragments of the invention may be immobilized together or next to each other (e.g. in the form of parallel lines). The antigens of the invention may also be combined with other antigens, e.g. antigens from other HIV-1 group O strains, or from HIV-1 group M or from HIV-2 strains.
The combination of different antigens in one single detection method as described above has certain advantages, such as:
achieving a higher test sensitivity: e.g. by combining several antigenic determinants from different HIV-strains, the total number of positively reacting sera originating from HIV-infected patients will be greater, and/or
enabling differentiation between individuals infected by different strains of HIV, more particularly enabling differentiation between HIV-1 group M, HIV-1 group O and HIV-2 infected patients.
The invention thus also relates to a solid support onto which the antigens of the invention, possibly in combination with other antigens, have been immobilized.
Another embodiment of the invention provides for a method for detecting the presence of HIV-1 nucleic acids, including HIV-1 group O nucleic acids, in a biological sample, comprising:
(I) possibly extracting the polynucleic acids contained in the sample,
(ii) possibly amplifying the HIV-1 polynucleic acids, including the HIV-1 group O polynucleic acids, with a suitable primer pair,
(iii) detecting the amplified nucleic acids, after hybridization with a probe as described above.
The expression xe2x80x9ca suitable primer pairxe2x80x9d refers to a pair of primers allowing the amplification of the target region to which the probes of the current invention hybridize. Depending on the application, the primer sequences may be chosen such that they amplify specifically the nucleic acids of the current invention, or, on the other hand, it may be preferred to obtain a more general amplification, e.g. of all or nearly all HIV-1 group O sequences, or of all or nearly all HIV-1 sequences, and even HIV-2 sequences.
In case a general amplification of HIV-1 group M and HIV-1 group O sequences is preferred, the following pair of primers may be used to amplify part of the gp41 region:
5xe2x80x2-GGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCG-3xe2x80x2 (SEQ ID NO 139), and
5xe2x80x2-TCTGAAACGACAGAGGTGAGTATCCCTGCCTAA-3xe2x80x2 (SEQ ID NO 140)).
In case a more specific amplification of the gp41 region of HIV-1 group O strains is preferred, the following pair of primers may be used:
5xe2x80x2-TGGATCCCACAGTGTACTGAAGGGTATAGTGCA-3xe2x80x2 (SEQ ID NO 141), and
5xe2x80x2-CATTTAGTTATGTCAAGCCAATTCCAAA-3xe2x80x2 (SEQ ID NO 142)).
The invention also relates to a method for genotyping HIV-1 or HIV-1 type O strains, comprising the following steps:
possibly extracting the nucleic acids from the sample,
amplifying the HIV-1 or HIV-1 type O nucleic acids using a suitable primer pair,
hybridizing the nucleic acids of the sample with at least one probe as described above,
detecting the hybrids formed,
inferring the presence of one or more HIV-1 or HIV-1 type O genotypes from the hybridization pattern obtained.
The term xe2x80x9cgenotypingxe2x80x9d refers to the typing of HIV-strains according to the sequence of their nucleic acids. Depending on the application, it may be the intention of a genotyping assay to differentiate between large groups of HIV-strains (e.g. HIV-1 group M; HIV-1 group O or HIV-2) or to subtype smaller entities (such as e.g. the clades withing HIV-1 group M (A to J)). Subtyping within HIV-1 group O may also be accomplished using the nucleic acids of the current invention.
Conditions allowing hybridization are known in the art and e.g. exemplified in Maniatis et al. (1982). However, according to the hybridization solution (SSC, SSPE, etc.), the probes used should be hybridized at their appropriate temperature in order to attain sufficient specificity (in some cases differences at the level of one nucleotide mutation are to be discriminated).
Amplification of nucleic acids present in a sample prior to detection in vitro may be accomplished by first extracting the nucleic acids present in the sample according to any of the techniques known in the art, and second, amplifying the target nucleic acid by any amplification method as specified above. In case of extraction of RNA, generation of cDNA is necessary; otherwise cDNA or genomic DNA is extracted.
The term xe2x80x9clabelledxe2x80x9d refers to the use of labelled nucleic acids. This may include the use of labelled nucleotides incorporated during the polymerase step of the amplification such as illustrated by Saiki et al. (1988) or Bej et al. (1990) or labelled primers, or by any other method known to the person skilled in the art. Labels may be isotopic (32P, 35S, etc.) or non-isotopic (biotin, digoxigenin, etc.).
Suitable assay methods for purposes of the present invention to detect hybrids formed between oligonucleotide probes according to the invention and the nucleic acid sequences in a sample may comprise any of the assay formats known in the art. For example, the detection can be accomplished using a dot blot format, the unlabelled amplified sample being bound to a membrane, the membrane being incubated with at least one labelled probe under suitable hybridization and wash conditions, and the presence of bound probe being monitored. Probes can be labelled with radioisotopes or with labels allowing chromogenic or chemiluminescent detection such as horse-radish peroxidase coupled probes.
An alternative is a xe2x80x9creversexe2x80x9d dot-blot format, in which the amplified sequence contains a label. In this format, the unlabelled oligonucleotide probes are bound to a solid support and exposed to the labelled sample under appropriate stringent hybridization and subsequent washing conditions. It is to be understood that also any other assay method which relies on the formation of a hybrid between the nucleic acids of the sample and the oligonucleotide probes according to the present invention may be used.
According to an advantageous embodiment, the process of detecting HIV-1 type O nucleic acids contained in a biological sample comprises the steps of contacting amplified copies of the nucleic acids present in the sample, with a solid support on which probes as defined above, have been previously immobilized. Preferably, the amplified nucleic acids are labelled in order to subsequently detect hybridization.
In a very specific embodiment, the probes have been immobilized on a membrane strip in the form of parallel lines. This type of reverse hybridization method is specified further as a Line Probe Assay (LiPA), and has been described more extensively in for example WO 94/12670.
The invention thus also relates to a solid support onto which the nucleic acids of the invention have been immobilized.
The invention also provides for a composition comprising at least one of the antigens or antigen fragments as above described, and/or at least one of the nucleic acids or nucleic acid fragments as above described, and/or an antibody as above described.
Examples of such compositions may be e.g. a diagnostic kit, an immunogenic composition, e.a.
In particular, the invention provides for a kit for the detection of the presence of an HIV-1 infection, comprising at least one of the antigens or antigen fragments as described above and/or at least one of the nucleic acids or nucleic acid fragments as described above and/or an antibody as described above.
More specifically, the current invention provides for a diagnostic kit for determining the presence of HIV-1 nucleic acids, including HIV-1 type O nucleic acids, in a biological sample, said kit comprising at least one nucleic acid fragment as described above. This nucleic acid fragment may be used as a primer or a probe in said kit.
In addition, the current invention provides for a kit for genotyping HIV-1 strains, including HIV-1 type O strains, in a biological sample, said kit comprising at least one nucleic acid fragment as described above. This nucleic acid fragment may be used as a primer or a probe in said kit.
Moreover, the present invention also provides for a kit for determining the presence of anti-HIV-1 type O) antibodies present in a biological sample, comprising at least one antigen or antigen fragment as described above.
In addition, the present invention provides for a kit for determining the presence of HIV-1 type O antigens present in a biological sample, comprising at least one antibody as described above.
The current invention also provides for a vaccine composition which provides protective immunity against HIV-1 infection, in particular against HIV-1 group O infection, comprising as an active principle at least one antigen or antigen fragment as described above, or at least one nucleic acid as described above, or a virus like particle (VLP) comprising at least one antigen or antigen fragment as described above, or an attenuated form of at least one of the HIV-1 type O strains as described above, said active principle being combined with a pharmaceutically acceptable carrier.
In a specific embodiment, polynucleic acid sequences coding for any of the antigens or antigen fragments as defined above, are used as a vaccine, either as naked DNA or as part of recombinant vectors. In this case, it is the aim that said nucleic acids are expressed into immunogenic protein/peptide and thus confer in vivo protection to the vaccinated host (e.g. Ulmer et al., 1993).
The active ingredients of such a vaccine composition may be administered orally, subcutaneously, conjunctivally, intramuscularly, intra nasally, or via any other route known in the art including for instance via the binding to carriers, via incorporation into liposomes, by adding adjuvants known in the art, etc.